EP2521735A2 - Diabody bivalent humain recombinant contre le virus de la rage et utilisation de celui-ci - Google Patents

Diabody bivalent humain recombinant contre le virus de la rage et utilisation de celui-ci

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Publication number
EP2521735A2
EP2521735A2 EP11708575.3A EP11708575A EP2521735A2 EP 2521735 A2 EP2521735 A2 EP 2521735A2 EP 11708575 A EP11708575 A EP 11708575A EP 2521735 A2 EP2521735 A2 EP 2521735A2
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EP
European Patent Office
Prior art keywords
diabody
seq
rabies virus
recombinant
recombinant human
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP11708575.3A
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German (de)
English (en)
Inventor
Venkata Nimmagadda Sridevi
Thirumeni Nagarajan
Dev Chandran
Alwar Villuppanoor Srinivasan
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Indian Immunologicals Ltd
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Indian Immunologicals Ltd
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Publication date
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Publication of EP2521735A2 publication Critical patent/EP2521735A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus

Definitions

  • the present invention is in the field of immunology in particular production of recombinant : antibody fragments.
  • the present invention specifically relates to production of recombinant human bivalent diabody protein against rabies virus.
  • Rabies is an invariably fatal viral infection of the nervous system of warm blooded animals including humans. It is transmitted by the bite of an infected animal, usually from a dog (Jackson, A.C., (2003). Rabies virus infection: an update. Journal for Neurovirology, 9; 253-258). It is estimated that every year 2.5-3 million people in India require post-exposure vaccination (Hemachudha T, Phuapradit P. (1997). Rabies. Current Opinion in Neurology. 10; 260-267). Potency determination of rabies vaccines involving the use of in vivo tests in mice (WHO, 1992) and in vitro methods based on estimation of rabies virus glycoprotein have been described (Perrin et al. (1990).
  • the first object of the present invention is to provide recombinant human bivalent diabody protein capable of recognizing rabies virus glycoprotein.
  • the second object of the present invention is to provide a method of production of the recombinant human bivalent diabody specific for rabies virus glycoprotein.
  • the third object of the present invention is to develop an ELISA for the quantification of rabies viral glycoprotein.
  • One aspect of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.
  • Another aspect of the present invention provides a polynucleotide encoding recombinant human bivalent diabody, wherein nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.
  • Figure 1 shows electrophoretic analysis of PCR amplified variable heavy chain (A), variable light chain (B) and the assembled diabody product (C). These gene sequences were amplified from total RNA isolated from a hetero-hybridoma secreting a fully human monoclonal antibody; Lane: M-Molecular weight marker, VH-Variable heavy chain, VL-Variable light chain and D-Assembled diabody product.
  • FIG. 2 shows SDS-PAGE analysis of purified bivalent diabody.
  • the purified protein was detected by staining with Coomassie brilliant blue and was determined to be of 27 KDa by running along with protein molecular weight marker.
  • Figure 3 shows western blot analysis of purified bivalent diabody fractions obtained after column chromatography. The blot was reacted with His probe specific for the Histidine tag of the bivalent diabody. A protein band of 27 kDa was detected.
  • Lanes Lane 1 -5, fractions obtained from column chromatography purification of bivalent diabody; Lane M: Prestained molecular weight marker.
  • Figure 4 illustrates an immuno- capture ELISA using the bivalent diabody.
  • the test sample B containing M5B4 (anti-rabies murine monoclonal antibody), PV Ag (Pasteur virus antigen), D06 (bivalent diabody) and His-Probe.
  • a and C are the negative control.
  • A is without diabody and C is without the PV Ag.
  • Figure 5 illustrates competitive ELISA using the bivalent diabody and the anti- rabies murine monoclonal antibody (M5B4).
  • A contains PV Ag, E. coli Lysate, M5B4 (anti-rabies murine monoclonal antibody), and anti mouse HRP.
  • B is without E. coli lysate.
  • Figure 6 illustrates the vector map showing the cloned bivalent diabody fragment.
  • Figure 7 shows the SDS-PAGE analysis of human monoclonal antibody secreted by heterohybridoma clones after affinity purification on protein A sepharose column. The antibodies are run along with BSA as standard.
  • Figure 8 illustrates the schematic representation of the constructed vector containing diabody gene cloned between EcoRl and Noil sites of pET 28a bacterial expression vector.
  • Figure 9 shows the specific binding of diabody to Rabies viral structural proteins transferred onto PVDF membrane by Immunoblot assay
  • Figure 10 shows the binding specificity of diabody by immunoreactivity assay
  • diabody used herein refers to an engineered antibody and/or antibody fragments that are bivalent, monospecific or bispecific molecules generated by dimerization of two variable heavy-variable light fragments.
  • bivalent used herein refers to an antibody and/or antibody fragment having two antigen binding sites capable of binding to two molecules of same or different antigens with great avidity.
  • the present invention provides a method for production of recombinant bivalent diabody against rabies virus.
  • the present invention further provides bivalent diabody rabies, composition comprising the bivalent diabody fragment and uses thereof.
  • the bivalent diabody fragment disclosed in the present invention recognizes rabies virus glycoprotein.
  • the bivalent diabody were constructed using gene sequences of a fully human monoclonal antibody secreted by a human X mouse heterohybridoma.
  • the present invention relates to the development of a bivalent diabody fragment constructed from a heterohybridoma (human X mouse) and having two antigen binding sites that is reactive against the glycoprotein of rabies virus and its use in the development of ELISA for the quantitation of rabies virus glycoprotein.
  • the present invention provides a method for production of recombinant bivalent diabody fragment construction from heterohybridoma and their use in the quantification of rabies glycoprotein.
  • Heterohybridoma was generated by immortalizing immune human B cells mediated by a human X mouse heteromyeloma (Champion et al. (2000).
  • the recombinant bivalent diabody was constructed from heterohybridoma and this recombinant diabody was used in the development of an ELISA for the quantitation of rabies virus glycoprotein in the vaccine manufacture.
  • DNA encoding variable domains of heavy and light chains were amplified separately by PCR and assembled into diabody with a short linker by splicing overlap extension polymerase chain reaction (SOE PCR).
  • SOE PCR splicing overlap extension polymerase chain reaction
  • Sequence of the recombinant diabody was verified and cloned into pET 28a vector and transformed into E. coli strain (BL21 -DE3) cells for soluble expression of diabody.
  • the soluble diabody protein was purified using immobilised metal affinity chromatography method. The purified protein was checked for their antigen binding activity using the rabies virus glycoprotein and was found to be nearly identical to the parental antibody.
  • the present invention further provides use of the recombinant diabody disclosed in the present invention for detection and quantification of rabies virus glycoprotein in the vaccine manufacture.
  • the recombinant human diabody which was constructed from heterohybridoma is one of the smallest recombinant bispecific antibodies consisting of two antigen binding sites devoid of constant regions and therefore provides high binding avidity and specificity to the target antigens similar to the parent antibody.
  • the recombinant bivalent diabody as disclosed in the present invention can be over expressed in bacteria and produced in large quantities at low cost to guarantee the supply of a consistent and well-characterized specific reagent to quantify rabies virus glycoprotein which can than be used to evaluate vaccine potency.
  • a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.
  • Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the rabies virus is selected from genotype (GT) 1 (GT1) (PV, Flury LEP, SAD, CVS- 1 1), GT4 ⁇ Duvenhage virus (DUV) ⁇ and GT7 ⁇ Zealandn bat lyssavirus (ABLV) ⁇ .
  • Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the diabody is a monoclonal antibody.
  • Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the diabody is a fully human monoclonal antibody.
  • Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus, wherein the diabody is monospecifc monoclonal antibody.
  • Another embodiment of the present invention provides a polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.
  • Another embodiment of the present invention provides a polynucleotide encoding recombinant human bivalent diabody, wherein nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25, wherein the polynucleotide encodes recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.
  • Yet another embodiment of the present invention provides a recombinant DNA expression cassette comprising the polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25, wherein the polynucleotide is operably linked to a promoter.
  • the present invention provides a recombinant vector comprising the recombinant DNA expression cassette comprising the polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25, wherein the polynucleotide is operably linked to a promoter.
  • Yet another embodiment of the present invention provides a recombinant host cell comprising the DNA expression cassette comprising the polynucleotide encoding recombinant human bivalent diabody, wherein the nucleotide sequence of the polynucleotide is as set forth in SEQ ID NO: 25.
  • Yet another embodiment of the present invention provides host cell selected from a group consisting of E. coli, yeast and CHO cells.
  • the present invention further provides a composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus and neutralizes the virus.
  • the present invention further provides a composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 and pharmaceutically acceptable carrier, wherein the diabody binds to rabies virus and neutralizes the virus.
  • the present invention also provides a vaccine composition
  • a vaccine composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 or the polynucleotide sequence as set forth in SEQ ID NO: 25.
  • the present invention also provides a vaccine composition comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.
  • Another embodiment of the present invention provides a therapeutic biological composition
  • a therapeutic biological composition comprising the recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 or the polynucleotide having the nucleotide sequence as set forth in SEQ ID NO: 25.
  • kits for estimation of rabies virus glycoprotein comprising a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26.
  • a method for treating rabies virus associated disease and/or disorders is also provided herein, wherein the method comprises administering an effective amount of the recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 to the subject in need thereof.
  • the present invention also provides use of the recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26 to the subject in need thereof for the preparation of medicament for the treatment of rabies associated disease or disorders.
  • Still another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody is produced in bacteria.
  • Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus glycoprotein.
  • Another embodiment of the present invention provides a recombinant human bivalent diabody having amino acid sequence as set forth in SEQ ID NO: 26, wherein the diabody binds to rabies virus glycoprotein G.
  • Still another embodiment of the present invention provides a test kit for estimation of rabies virus glycoprotein, wherein the kit comprises a recombinant human bivalent diabody encoded by the polynucleotide as set forth in SEQ ID NO: 25.
  • Various vaccine batches tested by diabody based IC-ELISA showed a good correlation with the NIH mouse potency studies as seen with MAb-M5B4 based IC-ELISA (Nagarajan et al 2006, WHO 1988).
  • Quantification of RV GP using diabody based IC-ELISA provides exact information of the natively folded RV GP antigen in vaccine preparations and in-process control samples which enables reliable estimation of RV GP.
  • diabody based IC-ELISA could replace the MAb based IC-ELISA due to better reagent stability and ease of production.
  • Rabies Virus Strain used in the present invention is the Pasteur virus (PV) strain described in literature.
  • Human X mouse heterohybridoma was generated by fusing immune human B cells with human X mouse heteromyeloma (Champion et al. (2000); The development of monoclonal human rabies virus-neutralizing antibodies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus exposure. Journal of Immunological Methods 235(1-2): 81 -90).
  • the heterohybridomas were developed by fusing primary immune peripheral blood B-cells and a heteromyeloma cell line, K6H6/B5 (Carroll et al. (1986); Mouse x human heterohybridomas as fusion partners with human B cell tumors.
  • the K6H6/B5 cell line was chosen because it has been successfully used to clone human anti-viral antibodies (Siemoneit et al. (1994). Isolation and epitope characterization of human monoclonal antibodies to hepatitis C virus core antigen. Hybridoma. 13(1): 9-13; Funaro et al. (1999). Identification of a 220-kDa membrane tumor-associated antigen by human anti-UK1 14 monoclonal antibodies selected from the immunoglobulin repertoire of a cancer patient. Experimental Cell Research 247(2): 441 -450).
  • the immune B cells isolated from a human subject vaccinated several times with the human rabies vaccine PVRV (Abhayrab, Human Biologicals Institute, Udhagamandalam) which incorporates PV strain of rabies virus which is known to provide broad coverage against street rabies virus (SRV) isolates (Badrane H, Bahloul C, Perrin P and Tordo N (2001). Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity, Journal of Virology 75; 3268-3276) were stimulated with pokeweed mitogen, before fusion. Using the hybridoma method, 8 new human IgG antibodies (huMabs) that bind the rabies virus glycoprotein were cloned.
  • PVRV Human Biologicals Institute, Udhagamandalam
  • the 8 new human IgG antibodies were characterized to determine their isotype, specificity and cross-reactivity.
  • Each of the huMabs was of gamma 1 ( ⁇ ) heavy chain and lambda ( ⁇ ) light chain isotype (Table 1 ).
  • Their specificity to RV was demonstrated by a Cell-ELISA using unfixed mock and RV infected cultured cells. All the huMabs showed reactivity to all the fixed RVs except the BHK-21 -adapted CVS strain (CVS- RV). None of the huMabs showed any reactivity to host cell protein (Table 2).
  • the huMabs bound specifically to native form of whole virus antigens as well as purified rabies virus glycoprotein and did not react with rabies virus nucleoprotein as evident from the results of Indirect ELISA (Table 1 ).
  • the huMabs recognized antigenic site III of rabies virus glycoprotein as determined by a competitive ELISA using a mouse Mab (Dl ) against antigenic site III as described elsewhere (Nagarajan et al. (2006), A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture, Biologicals. 34: 21 -27).
  • the huMabs have been tested for their ability to neutralize various street and fixed RVs both in vitro and in vivo.
  • mouse neutralization test MNT
  • the huMabs neutralized all the four Indian street rabies viruses (SRVs) of dog origin (108, 141 , 142, & 129) (Table 3).
  • All the 8 huMabs neutralized four fixed RV strains viz. Pasteur Virus (PV), Flury LEP, SAD and CVS- 1 1 ) while none of the huMabs neutralized BHK-21 adapted CVS RV when tested by rapid fluorescent focus inhibition test (RFFIT) (Table 4).
  • PV Pasteur Virus
  • RFFIT rapid fluorescent focus inhibition test
  • the clones were screened for the specific human Mab secretion by indirect ELISA employing purified inactivated rabies virus antigen (PV strain).
  • the human monoclonal antibody (Mabs) secreted by heterohybridoma clones have been affinity purified on protein A sepharose column.
  • the purified antibody was checked on gel ( Figure 7).
  • Lane 1 shows BSA as standard whose molecular weight is 66 KDa.
  • Lane 2-5 shows the purified R16E5 Human monoclonal antibody of molecular weight 160 KDa.
  • the main criterion for selecting four out of 8 clones was rabies virus neutralization spectrum as determined by RFFIT.
  • the huMabs were screened for the rabies whole virus antigen specific activity by indirect ELISA.
  • An ELISA plate was coated with zonal purified rabies whole virus antigen (1 : 100 dilution) in carbonate buffer overnight at 2-8°C. The plate was washed thrice with PBST and the unsaturated sites were blocked with 1% bovine gelatin.
  • the antigen coated on the solid phase was probed with huMabs and the presence of immune complex detected with goat anti-human IgG-peroxidase conjugate followed by the addition of TMB chromogenic substrate
  • the plate was read at 450 nm wavelength after stopping the colour reaction with 1.25M H 2 S0 . Growth medium and immune mouse serum were used as negative and positive controls respectively. The results are furnished in Table 1. It is clear from the results that the huMabs exhibited rabies whole virus antigen specific activity.
  • the huMabs were screened for the rabies virus specific activity by cell ELISA using rabies virus (PV strain) infected and mock infected VERO monolayer cell culture. The unsaturated sites were blocked using 1% bovine gelatin after fixing the cell sheet with 70% cold acetone. The plate was washed thrice with PBST and the fixed cell sheet was then was probed with huMabs. The presence of immune complex was detected with goat anti-human IgG-peroxidase conjugate followed by the addition of TMB chromogenic substrate. Growth medium and immune mouse serum were used as negative and positive controls respectively. The results are furnished in Table 2. It is clear from the results that the huMabs exhibited rabies virus specific activity essentially free from any cross-reactivity with the host cell proteins.
  • the huMabs were screened for their ability to neutralize fixed strain of rabies virus by RFFIT.
  • the huMabs were heat inactivated before setting up a neutralization reaction with 50 FFD 5 o of rabies virus (CVS-1 1 strain) by incubating at 37°C for 90 minutes.
  • the presence of unneutralized virus in the mixture after incubation was determined by seeding it along with an indicator cell line (MNA cell line) and incubated for 20 hours.
  • MNA cell line indicator cell line
  • the cell sheet was fixed with 70% cold acetone and probed with rabbit anti rabies virus nucleocapsid IgG-FITC conjugate to demonstrate the presence of rabies virus.
  • Human serum of known titer sourced from NIBSC, UK was used as the internal reference standard. The results are furnished in Table 4. It is clear from the results that the huMabs potentially exhibited rabies virus neutralizing activity.
  • the huMabs were screened for their ability to neutralize fixed and street rabies viruses by MNT.
  • the huMabs were heat inactivated before setting up a neutralization reaction with 50 LD50 of either fixed or street rabies virus by incubating at 37°C for 90 minutes.
  • the presence of unneutralized virus in the mixture after incubation was determined by intracerebral ly inoculating weaned mice and observed daily for the signs of rabies such as typical hind limb paralysis for a period of 21 days.
  • HRIG of known titer obtained from a commercial vendor was used as the internal reference standard. The results are furnished in table 3. It is clear from the results that the huMabs potentially exhibited the ability to neutralize fixed and street rabies viruses.
  • RNA was isolated from anti rabies heterohybridoma (R16E5) and DNA was synthesized by RT-PCR.
  • the nucleotide sequence of the amplified c-DNA is as set forth in SEQ ID NO: 25.
  • the RT-PCR amplified cDNA was used as template for amplification of variable domains of an antibody with universal primers having nucleotide sequence as set forth in SEQ ID NO: 1 to SEQ ID NO: 20).
  • the amplified variable domains were assembled to form diabody by splicing by overlap extension PCR and cloned in TOPO vector for sequence verification ( Figure 1A, Figure IB and Figure 1C).
  • HuVHla SEQ ID NO: 1
  • HuVH3a SEQ ID NO: 3
  • HuVH4a SEQ ID NO: 4
  • HuVH5a SEQ ID NO: 5
  • HuVH6a SEQ ID NO: 6
  • HuJHl-2 SEQ ID NO: 7
  • HuJH3 SEQ ID NO: 8
  • HuJH4-5 SEQ ID NO: 9
  • HuJH6 SEQ ID NO: J O
  • HuLAMl SEQ ID NO: 1 1
  • HuLAM2 SEQ ID NO: 12
  • HuLAM3a SEQ ID NO: 13
  • HuLAM3b SEQ ID NO: 14
  • HuLAM4 SEQ ID NO: 15
  • HuLAM5 SEQ ID NO: 16
  • HuJLAMl SEQ ID NO: 18
  • HuJLAM2-3 SEQ ID NO: 19
  • HuJLA 4-5 SEQ ID NO: 20
  • variable heavy chain (VH) and light chain (VL) were amplified from the cDNA using the universal human variable primers.
  • Human variable heavy chain (HuVH) forward primers and human joining heavy chain (HuJH) reverse primers were used to amplify variable heavy chain.
  • VH antibody variable heavy chain
  • V L variable light chain
  • the PCR reaction conditions are kept in the following order: 95°C x 5 minutes, 92 °C x 1 minute, 63 °C x 1 minute, 72 °C x 1 minute for 34 . cycles, and 72 °C x 10 minutes.
  • PCR program was arranged in the following order: 95 °C x 5 minutes, 92 °C x 1 minute, 63 °C x 1 minute, 72 °C x 1 minute for 14 cycles, and 72 °C x 10 minutes.
  • the PCR product obtained was used as a template and amplified with primers listed above as SEQ ID NO: 1 1 to SEQ ID NO: 17 as forward primer and SEQ ID NO: 7 to SEQ ID NO: 10 as reverse primer.
  • the PCR conditions followed are: 95 °C x 5 minutes, 92 °C x 1 minute, 63 °C x 1 minute, 72 °C x 1 minute for 34 cycles, and 72 °C x 10 minutes.
  • the resultant PCR product was amplified with the primers listed above as SEQ ID NO: 21 and SEQ ID NO: 22 to obtain the PCR product of 714 base pairs (SEQ ID NO: 25) for construction of diabody.
  • the amplified variable heavy and light chains were joined together with the polypeptide linker using SOE and the resulted PCR product of 714 base pairs (SEQ ID NO: 25) was cloned into TOPO-TA vector. Positive clones which showed release of 714 base pairs (bps) product after enzymatic analysis of the plasmids were sequenced and blasted the sequence by using NCBI blast search showed the presence of 363 bps of variable heavy chain, 327 bps of variable light chain and 24 bps linker region which forms a diabody of 714 bps having the polynucleotide as set forth in SEQ ID NO: 25.
  • the resultant PCR product of 714 base pairs (SEQ ID NO: 25) was further amplified using the primers as set forth in SEQ ID NO: 23 and SEQ ID NO: 24 to add the restriction site for cloning into pET vector.
  • Primer 2 SEQ ID NO: 24
  • the diabody fragment having polynucleotide as set forth in SEQ ID NO: 25 was cloned between EcoRl and Noil sites of pET 28a bacterial expression vector.
  • the vector pET 28a and the insert referred to diabody of size 714 bps was digested with EcoRl and Noil, respectively by incubating at 37°C for 12 hours.
  • the digested products was purified using the kit provided by QIAGEN and kept for various ratios of vector to insert (i.e., 1 :3 and 1 :6) blunt end ligation and incubated at 22°C for 2 hours.
  • the ligated product was incubated for further 20 minutes at 65°C in order to inactive the enzyme.
  • the pET 28a vectors carry an N-terminal His tag/thrombin/T7 Tag configuration plus an optimal C-terminal His tag sequence.
  • T7 promotor and T7 terminator are situated used to sequence the single stranded insert DNA fragment of the diabody. The insert was verified by sequencing the clone from vector backbone using T7 promotor and T7 terminator primers. The obtained 714 base pairs sequence is as set forth in SEQ ID NO: 25.
  • Overnight grown XL-Blue strain culture was sub-cultured and grown at 37°C, shaking until the OD of the culture reaches to 0.6 at 600nm.
  • the culture was harvested by centrifuging at 5000 x rpm for 10 minutes at 4°C and resuspended in ice-cold O. lmM CaCl 2 and incubated overnight on the ice, before proceeding for transformation.
  • the chemically competent XL-Blue cells were incubated with plasmid DNA for 30 minutes on ice.
  • the cells were given heat shock at 42°C for 90 seconds and immediately placed in ice for 2 minutes before the media was added to cells.
  • the cells were incubated for one hour at 37°C for recovery and plated on semi-solid media containing 50mg/ml of kanamycin.
  • the plates were incubated for overnight and screened for positive clones by isolating the plasmids and subjected to digestion with EcoRl and No/I.
  • the positive clones were sequence verified before the plasmid was transformed into BL-21 DE3 cells of E .coli for soluble expression of the antibody gene.
  • the recombinant diabody fragment having polynucleotide sequence as set forth in SEQ ID NO: 25 was expressed in E. coli for production of recombinant human monoclonal diabody protein having polypeptide sequence as set forth in SEQ ID NO: 26. Further, the diabody protein was purified from the soluble fraction of the lysate using Immobilized Metal Affinity Chromatography (IMAC).
  • IMAC Immobilized Metal Affinity Chromatography
  • the purified recombinant human diabody protein was analyzed and a band of 27 KDa size was visualized on 12% reducing SDS-PAGE after staining with Coomassie brilliant blue ( Figure 2) and detected with His-Probe in western blotting ( Figure 3) Yield of the affinity purified protein was quantified using Bicinchoninic Acid kit and found to be on an average of 5mg/10L.
  • Immuno capture ELISA was performed to check the binding affinity of the recombinant human bivalent diabody protein.
  • a micro titer plate was coated with mouse monoclonal antibody (Mab) M5B4 (l OOng/well) in carbonate buffer by incubating over night at 4°C. The plate was washed thrice with PBST and the unbound sites in the wells were blocked with 1% bovine gelatin. Purified rabies virus glycoprotein (Pasteur virus strain) was added in different dilutions and allowed to react with M5B4. Diabody protein tagged with Histidine was added and allowed to react with captured antigen. The affinity binding of the diabody with rabies virus glycoprotein was detected by adding His-Probe and a chromogenic substrate TMB. The plate was read at 450nm after the reaction was stopped with 1.25M H 2 So 4 ( Figure 4) .
  • IC-ELISA was performed to quantify the Rabies Virus Glyco Protein (RV GP) content in rabies vaccine formulations, according to the method described by Nagarajan et al. ⁇ Nagarajan, T., G. S. Reddy, B. Mohana Subramanian, S. Rajalakshmi, D.Thiagarajan, N. Tordo, C. Jallet, and V. A. Srinivasan. 2006. A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture. Biologicals 34:21-27. ⁇ , with a few modifications wherein the diabody (450 ng/well) was used for detection.
  • RV GP Rabies Virus Glyco Protein
  • ELISA plate was coated with mouse monoclonal antibody (MAb) M5B4 overnight at 4°C and the un-reacted sites blocked with 1% bovine gelatin.
  • the test vaccines and an internal reference standard (IRS) vaccine of known RV GP were subjected to 8 serial 2 fold dilutions in PBS-T.
  • the RV GP trapped by MAb M5B4 was detected using the diabody followed by the addition of anti-1 His Probe.
  • the plate was developed with TMB at room temperature for 10 minutes.
  • the reaction was stopped by addition of 1.25M H2S04 and the absorbance was measured at 450 nm wave length using a micro titre plate reader (BIO-TEK, USA).
  • the assay was performed in triplicate.
  • the RV GP content was also estimated by MAb-M5B4 IC- ELISA previously described b Nagarajan et al. 2006, using reference standard vaccine.
  • the RV GP content was estimated using the formula
  • NIH potency test was carried out on the different rabies vaccine formulations in mice using standard procedures (Wilber, L. A., and M. F. A. Aubert. 1996. The NIH test for potency, p.360-368. In F. X. Meslin, M. M. Kaplan, and H. Koprowski (ed.), Laboratory techniques in rabies, 4th ed. WHO, Geneva, Switzerland.).
  • PV GP was estimated in 65 batches of experimental human rabies vaccine preparations using the IC-ELISA and the estimates were compared to the NIH potency values of those respective batches.
  • ANOVA with Regression Through the Origin (RTO) model was performed to compare the potency estimates derived by M5B4-D06 IC-ELSIA and MAb-M5B4 IC-ELISA previously described by Nagarajan et al., (2006).
  • Regression analysis was performed using the data analysis program in Microsoft Excel 2003 to compare the potency estimates derived by M5B4D06 IC-ELSIA and MAb-M5B4 IC-1 ELISA with the NIH estimate.
  • the adjusted R2 value obtained was 0.902 and equation for predicted potency values for M5B4-D06 based IC-ELISA and MAb-M5B4 IC-ELISA were 0.565 lx and 0.8044x respectively, where x is the estimate of RV GP by the IC-ELISA in ⁇ g ( Figure 1 1a and l i b).
  • ANOVA results showed the estimates by the two methods compared differed highly significantly (PO.001 ) while the predicted potencies by the two test did not differ significantly (P>0.05).
  • mice Mab The constant amount of mouse Mab (M5B4) was added to each well containing diabody and E.coli lysate and the plates were incubated at 37°C for 1 hour. The plate was washed with PBST as mentioned above. Goat anti-mouse IgG HRP conjugate with the working dilution of 1 :5000 (as recommended by the manufacturer) was added to each well and the plates were incubated further at 37°C for 1 hour. The plate was washed five times with PBST before chromogenic substrate TMB was added. TMB substrate was prepared by dissolving one TMB tablet and 3 ⁇ ) of 3 ⁇ 4(1 ⁇ 4 in citrate buffer and an aliquot of ⁇ ⁇ added to each well. The reaction was stopped by adding ⁇ ⁇ of ] .25 M H2SO4 to each well and plate was read at 450 nm ( Figure 5).
  • CVS- 1 1 essentially as described by Smith et al. ( 1996).
  • Table 3 Survivorship of Swiss albino mice subjected to mouse neutralization test b to demonstrate the ability of human monoclonal antibodies to neutralize various fixed and street rabies viruses in vivo
  • mice Female Swiss albino mice (3-4 weeks old) were intracranialy inoculated with 30 ⁇ of (50 MlCLD 50 /30 ⁇ ) either rabies virus infected culture fluid or brain homogenates from naturally infected rabid stray dogs. The inoculated mice were observed daily for symptoms typical of rabies for 21 days. The results are expressed as per centage of mice that survived after 21 days of observation.
  • Table 5 Neutralization of rabies-related viruses by anti-rabies virus human monoclonal antibodies
  • VNA titer was determined by RFFIT at CDC, Atlanta, essentially as described by Smith et al. (1996).
  • RFFIT rapid fluorescent focus inhibition test
  • Partially purified buMAb preparation was used.
  • SEQ ID NO: 26 Amino acid sequence of diabody fragment (238 a. a.)

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Abstract

La présente invention concerne un diabody bivalent humain recombinant contre le virus de la rage qui est capable de reconnaître la glycoprotéine du virus de la rage et de neutraliser le virus de la rage, ainsi qu'un procédé de production de celui-ci. La présente invention concerne en outre un polynucléotide qui code le diabody bivalent recombinant. Le diabody bivalent divulgué dans la présente invention peut également être utilisé pour quantifier la glycoprotéine du virus de la rage afin d'évaluer la qualité du vaccin et de prédire l'activité thérapeutique du vaccin.
EP11708575.3A 2010-01-04 2011-01-03 Diabody bivalent humain recombinant contre le virus de la rage et utilisation de celui-ci Ceased EP2521735A2 (fr)

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JP5868549B2 (ja) 2012-05-24 2016-02-24 マウントゲイト グループ リミテッド 狂犬病感染の予防および治療に関する組成物および方法
WO2014059434A1 (fr) * 2012-10-12 2014-04-17 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Essai à des fins d'analyse de glycoprotéines de virus rabique
PL3220947T3 (pl) * 2014-11-18 2021-05-31 Humabs Biomed S.A. Przeciwciała silnie neutralizujące wirusa wścieklizny i inne lyssawirusy oraz ich zastosowanie
US11773155B2 (en) * 2018-08-09 2023-10-03 Beijing Wisdomab Biotechnology Co., Ltd Bispecific antibody against rabies virus, and application thereof
CN111999497B (zh) * 2020-08-24 2023-08-08 中牧实业股份有限公司 检测狂犬病毒糖蛋白抗原的酶联免疫试剂盒及其应用

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